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P G Layer

Publications and source records attributed to P G Layer.

72 records · Page 4Linked to original sources
PubMed1988

The pigmented epithelium sustains cell growth and tissue differentiation of chicken retinal explants in vitro.

Embryonic retinae from 5-6-day-old chicks (E5-E6) were cut into stripes either in close contact with (RPE stripes) or in absence of the neighboring retinal pigmented epithelium (R stripes). The stripes were explanted and cultivated in vitro for up to 6 days, during which time they show the following differences in their characteristics of growth and differentiation. Compared with R stripes, RPE stripes morphologically showed a significant increase in size during the first 2 days in culture. Using E5 tissue, this is also demonstrated by a higher rate of cell proliferation (as measured by uptake of radioactive thymidine as well as by DNA contents). In contrast, R stripes after two days in culture show a much stronger neurite growth. After longer periods of culturing (5-6 days) we can show by cholinesterase histochemistry (AChE and BChE) and by PNA-lectin binding that the RPE stripes have started to form all major layers of the in vivo retina, whereas R stripes remain unstratified and start to degenerate earlier. We conclude that the pigment epithelium might exert a specific stimulus on growth and tissue differentiation of the neural retina not only during in vitro, but possibly also during in vivo development. The in vitro methods introduced here could become useful model systems to further investigate the significance of the RPE for developmental, regenerative and even adult processes of the neural retina. Their future applicability in ophthalmologic research is briefly discussed.

Animals↗
PubMed1988

Sequential activation of butyrylcholinesterase in rostral half somites and acetylcholinesterase in motoneurones and myotomes preceding growth of motor axons.

By applying double-staining procedures that combine cholinesterase histochemistry (acetyl- and butyrylcholinesterase, respectively) as indicators of neuronal and myotomal tissue differentiation on longitudinal sections, together with detection of motor axons with antibodies to G4 antigen, we here describe the spatiotemporal expression of all components of the segmental motor units along the trunk of chicken embryos between stages 16-20. In particular, BChE expression is spatially elevated on the rostral part of the differentiating somite. About 2-3 somites more rostrally (and thus developmentally later), AChE is expressed almost simultaneously in a nonsegmented fashion in neuronal cell bodies of the ventral horn and in the corresponding dermomyotomes. There it is first detectable in a rostromedial sector. With a delay (4-6 somites compared with AChE in motoneurones), motor axons begin to grow exclusively through the BChE-rich sclerotomal space towards the AChE-activated myotome anlage. On motor axons, AChE detection is significantly retarded. We conclude that the rostrocaudal segmental asymmetry is not restricted to the sclerotomes (which other authors have described before by using different markers), but it extends into the dermomyotome, in which cholinesterases introduce an early subdivision. Hence, the entire process of first myotome differentiation, motor axon growth and establishment of first target contacts are taking place within the rostral half somite. We suggest that both cholinesterases might be involved in processes of motor unit differentiation and fibre guidance.

Acetylcholinesterase↗
PubMed1987

Cholinesterases and cell proliferation in "nonstratified" and "stratified" cell aggregates from chicken retina and tectum.

Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cell-to-cell relationships ("sorting out"), but these systems never form highly laminated aggregates ("nonstratified" R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly "stratified" aggregates ("RPE-aggregates", see Vollmer et al. 1984). The present comparative study of "stratified" and "nonstratified" aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates. During early aggregate formation, in both "stratified" and "nonstratified" aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2-3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE- and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1987

Spatiotemporal relationship of embryonic cholinesterases with cell proliferation in chicken brain and eye.

Close relationships between acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, true cholinesterase, EC, 3.1.1.7) and butyrylcholinesterase (BtChoEase, acylcholine acylhydrolase, pseudocholinesterase, EC, 3.1.1.8) with cell proliferation were observed in the early chicken brain. These include the following: BtChoEase is transiently accumulating in patchy fashion on the ventricular side of the neuroepithelium shortly before AcChoEase appears in cell bodies along the opposing mantle layer. The amount of BtChoEase in retina and brain is greatest in the early phase (E3-E5, or incubation periods of 3-5 days); in retina it decreases about 2 days later than in brain. However, AcChoEase expression increases with time, in inverse order to that of BtChoEase. In both tissues decrease of cell proliferation is closely followed by decrease in BtChoEase. A double-labeling technique of cholinesterase staining together with [3H]thymidine autoradiography reveals proliferation zones that are diffusely stained by BtChoEase but not by AcChoEase. Patches intensely stained for BtChoEase accompany clusters of cells in final stages of mitosis on their way to the differentiation zone, where they begin expressing AcChoEase. By applying different thymidine pulses, we identify an 11-hr lag from the last thymidine-uptake to full AcChoEase expression. (iv) These findings are confirmed by studying lens development, where areas of proliferation and differentiation are well separated. The spatiotemporal pattern of the transition of neuroblasts from a proliferating into a differentiating state correlates with the expression of BtChoEase just before and during mitosis and that of AcChoEase about 11 hr after mitosis. Thus cholinesterases could be involved in the regulation of this transition.

Acetylcholinesterase↗
PubMed1987

Quantitative development and molecular forms of acetyl- and butyrylcholinesterase during morphogenesis and synaptogenesis of chick brain and retina.

The embryonic development of total specific activities as well as of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and of butyrylcholinesterase (BChE, EC 3.1.1.8) have been studied in the chick brain. A comparison of the development in different brain parts shows that cholinesterases first develop in diencephalon, then in tectum and telencephalon; cholinesterase development in retina is delayed by about 2-3 days; and the development in rhombencephalon [not studied until embryonic day 6 (E6)] and cerebellum is last. Both enzymes show complex and independent developmental patterns. During the early period (E3-E7) first BChE expresses high specific activities that decline rapidly, but in contrast AChE increases more or less constantly with a short temporal delay. Thereafter the developmental courses approach a late phase (E14-E20), during which AChE reaches very high specific activities and BChE follows at much lower but about parallel levels. By extraction of tissues from brain and retina in high salt plus 1% Triton X-100, we find that both cholinesterases are present in two major molecular forms, AChE sedimenting at 5.9S and 11.6S (corresponding to G2 and G4 globular forms) and BChE at 2.9S and 10.3S (G1 and G4, globular). During development there is a continuous increase of G4 over G2 AChE, the G4 form reaching 80% in brain but only 30% in retina. The proportion of G1 BChE in brain remains almost constant at 55%, but in retina there is a drastic shift from 65% G1 before E5 to 70% G4 form at E7.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1986

Reaggregation of chick retinal and mixtures of retinal and pigment epithelial cells: the degree of laminar organization is dependent on age.

We report here a striking age dependency for tissue reconstruction capacity in rotating cultures of chick retinal cells, in particular in mixtures of retinal and pigment epithelial cells. The comparison of sections after acetylcholinesterase histochemistry delivered the following results. In aggregates from retinal cells, sorting out is clearly observable, but only a minor age-dependent effect is detectable, e.g. the laminar order of aggregates derived from 6-day-old cells is increased compared to aggregates from other stages. In aggregates from mixtures of retinal and pigment epithelial cells, the degree of laminar order is highest with cells from 5-day-old embryos and decreases significantly at older ages. The highest number of aggregates with the reconstruction of all main retinal layers was obtained by the reaggregation of retinal cells of embryonic day 5 (25%). To achieve laminar order in aggregates, at all ages, the addition of pigment epithelial cells seems to be a prerequisite.

Acetylcholinesterase↗
PubMed1986

Inhibition of cell proliferation by cytosine-arabinoside and its interference with spatial and temporal differentiation patterns in the chick retina.

Incubation of chick embryos with 200 nmoles/egg of cytosine arabinoside (AraC) completely inhibits cell proliferation in the embryo. At an age older than embryonic day 4 (E4) more than 90% of the embryos survive this treatment. The drug induces various malformations; in particular the retina is heavily affected. This simple method offers the possibility to study the effect of a more or less decreased cell production on processes of further differentiation and histogenesis of retinal tissue. The following results are obtained: In spite of the inhibition of cell proliferation in the retina by AraC an abnormally thick basal lamina is found and the expansion of the eye still proceeds, indicating that eye growth is not only dependent on retinal cell numbers. Stereotyped malformations of retinal histogenesis are induced and categorized into three groups: in addition to areas of normal structure cells are found arranged in rosettes and in half-rosettes sometimes linked by areas of undefined transient cell arrangements. The results point to a strong tendency of a severely diminished cell population to form regularly laminated retinal-like structures as long as a minimal ratio of cell types is given. The spatio-temporal appearance of the type of retinal malformation in a given retinal area is dependent on the time of AraC exposure and thus on the degree of differentiation reached at a "spatio-temporal spot": Full rosettes develop at earlier, and half-rosettes at later stages of AraC interference. Furthermore, deformities first appear on temporal and ventral sides. Thus, the establishment of these malformations follows and reflects the normal sequence of differentiation within the retina. Cells within rosettes organize in specific layers and start to differentiate normally. This shows that earlier formed cells are not dependent on the influence of factors derived from cells that are formed later for their proper differentiation.

Animals↗
PubMed1986

Lucifer Yellow stains a subpopulation of chicken retinoblasts preferentially 3 h after their last round of DNA synthesis.

Observations of histological preparations of E4-E6 retinae suggested to us that Lucifer Yellow (LY) uptake occurs during a specific phase of the mitotic cell cycle. We examined this phenomenon more closely in intact E6 retinae. If we first exposed the tissues to a 20-min pulse of tritiated thymidine followed by LY-staining at different intervals, a distinct peak of double-labelled cells was found 3 h after exposure of the tissue to thymidine. In contrast, if staining with LY is followed by thymidine pulses, only few double-labelled cells are found. We conclude that a subpopulation of young neurons takes up LY specifically after their last round of DNA-synthesis.

Animals↗
PubMed1986

An in vitro model of proliferation and differentiation of the chick retina: coaggregates of retinal and pigment epithelial cells.

Pigment epithelial (PE) cells exert a pronounced organizing effect when added to embryonic day (E) 5-6 chick retinal cells in a reaggregation system such that after a period of 14-21 d of culture, the main layers of an intact E10-E14 retina are reconstructed (Vollmer et al., 1984). In the present study we investigated the time course of the formation of retina-like structures in retinal-pigment epithelial aggregates, in particular, the fate of the PE cells and their influence on processes of differentiation within the aggregates. PE cells first form a core in the center of the aggregates and migrate to the periphery at later stages. The PE core affects the organization of proliferation and differentiation, phenomena that were monitored using 3H-thymidine autoradiography and AChE histochemistry, respectively. A double-staining procedure combining both techniques on the same section is described. Soon after aggregation, a matrix zone and a zone of differentiated cells are formed. At later steps, proliferation becomes gradually restricted to a narrow band within the aggregates comparable to the in vivo situation. The spatiotemporal pattern of withdrawal from mitosis resembles that of the in vivo retina. Proliferation in aggregates is sustained over a longer period with PE, as compared with aggregates formed by retinal cells alone. AChE staining in aggregates in the presence of PE shows a layered appearance, while there is only crude sorting-out of labeled and unlabeled cells in aggregates composed of retinal cells only. The basis of PE cell action as well as the relevance of this in vitro system for understanding normal eye development are briefly discussed.

Animals↗
PubMed1984

Reaggregation of embryonic chick retina cells: pigment epithelial cells induce a high order of stratification.

We report here that, in comparison to aggregates from retinal cells alone, addition of pigmented epithelial cells to retinal cells in rotary culture results in a pronounced increase of spatial order. A particularly high level of organization is found in about 15-20% of the aggregates. In these 'retinoids' the main layers characteristic of developing in vivo retinae can be distinguished in correct sequential arrangement on the basis of morphological criteria and by using acetylcholinesterase histochemistry [5, 6, 15], peanut agglutinin-lectin binding [11] and Lucifer Yellow staining [7-9].

Acetylcholinesterase↗
PubMed1984

Spatio-temporal patterns of differentiation of whole heads of the embryonic chick as revealed by binding of a FITC-coupled peanut-agglutinin (FITC-PNA).

The spatio-temporal expression of specific binding sites for FITC-labeled peanut agglutinin (Arachis hypogaea) in frozen sections of whole embryonic heads of the chick between embryonic day 2 (E2) and embryonic day 8 (E8) is described. PNA-binding sites are present already at E2 on the external limiting membrane and the ventricular boundary of the neural wall. With progressing differentiation fluorescent bands are formed from the outside of the neural wall radiating inside, forming second layers inside the CNS at later stages. The visual system (retina and tectum) shows remarkably little PNA-binding activity until about E7/8, a time when binding sites in the other brain areas are already most abundant. In the telencephalon a fuzzy distribution of fluorescence is found. A possible significance of this finding for fiber guidance from the retina to the tectum is briefly discussed. Local high activities can be observed in different parts of the brain. Intense bands of PNA-fluorescence on parts of the epidermis, along the eye cups, around the fore- and hindbrain and in the medial part of the mesenchyme delineate early biochemical compartments foreshadowing the succeeding ossification.

Animals↗
PubMed1983

Selective uptake of Lucifer Yellow into different cell populations of the developing chicken retina.

Whole chick embryo eyes at different stages of development were incubated in a solution of Lucifer Yellow (LY) and examined in frozen sections. Different cell populations selectively take up the dye during the course of development: from embryonic day 4 - day 8 of incubation (E4-E8) only a subpopulation of neuroepithelial cells are stained, which at E7-E8 are located mainly near the periphery between the prospective ora serrata and the outer edge of the retina. This staining disappears after E8. A second phase starts at E10, with a weak but specific staining of a small population of cells in the outer nuclear layer which becomes less pronounced by E12. Starting around E10.5 a bright staining of cells in the innermost part of the inner nuclear layer can be observed. Their size, shape and location suggest that most of them represent a subclass of amacrine cells (including all 'displaced amacrine cells'). Beginning at E15, the staining pattern becomes more complex. A single cell analysis of the percentage of LY-stained cells which are stained either before or after papain-treatment and dissociation of the intact tissue confirms and extends the histological findings.

Animals↗
PubMed1983

Binding and degradation of nerve growth factor by PC12 pheochromocytoma cells.

The specific binding of various concentrations of 125I-labeled nerve growth factor (NGF) to PC12 cells at 37 degrees C reached maxima after 90 min and then declined to 25% of maximal binding after 10 h. Decreased binding was accompanied by degradation of 125I-NGF and the appearance of acid-soluble biologically inactive 125I (mainly 125I-monoiodotyrosine) in the medium as well as a decrease in the number of surface NGF receptors. The time-dependent decrease in binding and the degradation of 125I-NGF were inhibited by low temperature and the lysosomotropic agent chloroquine while degradation was inhibited by metabolic energy inhibitors in the absence of glucose. Chloroquine also produced an increase in the accumulation of 125I-NGF which was not readily removed from the cells. These data suggest that 125I-NGF bound to PC12 cells is efficiently internalized by receptor-mediated endocytosis and degraded by the lysosomes. It appears from other data that this process does not produce the intracellular signals regulating neurite outgrowth.

Adrenal Gland Neoplasms↗
PubMed1983

Binding of FITC-coupled peanut-agglutinin (FITC-PNA) to embryonic chicken retinas reveals developmental spatio-temporal patterns.

FITC-labeled peanut agglutinin (PNA), a lectin from peanut (Arachis hypogaea) which binds specifically to D-galactose, was used as a probe to trace the distribution of PNA-lectin receptors during early development of chick retina. We found that specific fluorescence within the inner plexiform layer (IPL) and the outer plexiform layer (OPL) appears at different developmental stages. The specific fluorescence of the IPL first appears near the central area of the ventro-posterior quadrant of the 7-day retina, then extends progressively towards the periphery, reaching the ora serrata earlier on the posterior than on the anterior side. The development of the specific fluorescence of the OPL follows the same scheme, but it appears later (starting at day 9) than that of the IPL and it is less pronounced. The spatio-temporal patterns of the expression of the lectin receptors show relationships to those of withdrawal of proliferating cells from mitosis, and of other events related to differentiation.

Animals↗
PubMed1983

Asymmetrical developmental pattern of uptake of Lucifer Yellow into amacrine cells in the embryonic chick retina.

Whole chick embryo eyes between embryonic day 10.5 and 12 (E10.5-E12) were incubated in a solution of Lucifer Yellow and examined in frozen sections. Starting around day 10.5, brightly stained cells were observed in the innermost part of the inner nuclear layer. Their size, shape and location suggest that most of them represent a subclass of amacrine cells. A distinct spatio-temporal development of Lucifer Yellow-staining of this subpopulation of cells within the inner nuclear layer between E10.5 and E12 is revealed in detail using 3-dimensional models of Lucifer Yellow-stained eyes. The staining can be first observed at a specific location at the ventro-posterior side not far from the fundus. It then spreads radially in a complex pattern reaching the ora serrata first on the ventro-posterior side, then sequentially reaching into the dorso-posterior, the ventro-anterior and finally the dorso-anterior quadrants of the retina. Our results suggest that a horizontal (posterior-anterior) axis and a vertical (ventro-dorsal) axis function as a coordinate system during differentiation of the tissue. We conclude that there are precise spatio-temporal patterns of Lucifer Yellow-staining which most probably reflect spatio-temporal patterns of cell differentiation within the chicken retina. The correlation of these findings with other data on spatio-temporal patterns during differentiation of the chicken retina is discussed.

Animals↗
PubMed1983

Comparative localization of acetylcholinesterase and pseudocholinesterase during morphogenesis of the chicken brain.

The histochemical localization of specific acetylcholinesterase (AcChoEase) and nonspecific cholinesterase (BtChoEase) is described during the early morphogenesis of the whole chicken head with main emphasis on the visual system. It is found that: (i) Expression of AcChoEase is an early differentiation event in the entire brain. Its pattern of first appearance on the external part of the neuroepithelium correlates with the general spatio-temporal pattern of differentiation. AcChoEase thus represents an early differentiation marker. (ii) The late pattern of AcChoEase (at E18), reflecting at least partially the distribution of synaptic AcChoEase shows no direct correlation to the distributions found at early stages when synapses are not yet formed. This argues for a nonsynaptic function of the early appearing AcChoEase. (iii) BtChoEase in early nervous tissue is diffusely localized on the ventricular side of the neural tube. At later stages it becomes concentrated on the ependymal layer as well as along fibers reaching from this inner layer outwards. Minor activities appear in specific external layers of tectum and retina. (iv) During the course of differentiation the enzymes express pronounced graded distributions within the areas where they are detectable. (v) Mesenchymal and epidermal BtChoEase is abundant in the entire head. Prominent amounts of activity are expressed on the rostral epidermis, along the eye cup next to the sclera, and on the rostro-dorsal surrounding of the optic nerves. The results are discussed in the light of possible morphogenetic functions of these enzymes.

Acetylcholinesterase↗
PubMed1982

Lucifer yellow stains displaced amacrine cells of the chicken retina during embryonic development.

We have used Lucifer Yellow for histological tracing of displaced amacrine cells within the ganglion cell layer (GCL) during the embryonic development of the chicken retina. Incubating whole eyes in the dye leads to bright staining of all displaced amacrine cells, whereas ganglion cells and glial cells are not stained. A subpopulation of cells of the inner part of the inner nuclear layer (INL) are also stained (for further details see ref. 13). Kainic acid, which is known to interfere with and kill amacrine cell systems, blocks the staining of these cells fully. This in addition to histological evidence confirms that the LY-stained cells in the GCL are displaced amacrine cells. Of the cells in the GCL, 23% (+/- 3%) are of the displaced amacrine type. Further, we find that the cytoarchitectural arrangement of these cells changes significantly during development.

Animals↗